Phase I Metabolism of Tiletamine in Human Liver Microsomes and Its Detection in Human Specimens (Urine and Hair)

ABSTRACT As tiletamine emerges as a ketamine substitute, its detection in complex human matrices such as wastewater and hair remains challenging owing to the lack of specific biomarkers and targeted methods. This research addresses this gap by systematically identifying novel metabolites that can serve as definitive markers. Using ultra‐high‐performance liquid chromatography–high‐resolution mass spectrometry four metabolites (MeT1–MeT4) were characterized from in vitro and authentic samples, three of which (MeT2‐MeT4) are newly identified. Subsequent targeted screening of 469 clinical samples (433 hair and 36 urine samples) using a validated high‐performance liquid chromatography‐tandem mass spectrometry method revealed a distinct exposure profile: 33 hair samples were positive for tiletamine, with a consistent metabolite abundance order of MeT2 > MeT3 > MeT1 > MeT4. The parent compound was undetectable in urine and the possible metabolite MeT4 was present in only one urine sample, which corresponded to a hair sample positive for tiletamine. Overall, these findings underscore the necessity of monitoring metabolites. Given its high relative abundance, MeT2 is proposed as a robust and characteristic biomarker providing a critical reference for future wastewater‐based epidemiology and forensic hair testing for tiletamine.